Construction of vof16 gene knockout rat with CRISPR/Cas9

Objective: This study aimed to generate a vof16 gene knockout rat model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) technology, so as to explore the biological functions of vof16. Methods: The CRISPR/Cas9 system was employed to target the vof16 gene in fertilized Sprague-Dawley (SD) rat embryos. After microinjection of Cas9 mRNA and single guide RNAs (sgRNA) into the pronuclei, the modified embryos were implanted into pseudopregnant females. Offspring were genotyped and confirmed through polymerase chain reaction (PCR), agarose gel electrophoresis and sequencing. Results: Successfully edited vof16 knockout rats were produced, with targeted deletions validated through PCR, gel electrophoresis and sequencing. Agarose gel electrophoresis results showed specific deletions in the vof16 gene fragment of knockout rats, significantly differing from wild-type rats. Besides, the gene knockout rats exhibited stable genetic characteristics, providing reliable experimental materials for subsequent functional studies. Conclusion: The establishment of a vof16 gene knockout rat model demonstrates the effectiveness of CRISPR/Cas9 in generating precise genetic modifications. This model provides a valuable tool for studying the physiological and pathological roles of vof16, potentially offering new insights into related disease mechanisms and therapeutic strategies.